This application addresses broad Challenge Area (03) Biomarkers Discovery and Validation and specific Challenge Topic, 03-HL-101: Identify and validate clinically relevant, quantifiable biomarkers of diagnostic and therapeutic responses for blood, vascular, cardiac and respiratory dysfunction. The future of stem cell transplantation is dependent on noninvasive biomarkers to characterize cells and stem cell aggregates prior to transplantation. Characterization is needed minimally to distinguish stem cells from differentiated cells and ideally to predict the cells best poised to contribute to a specific tissue type. The noninvasive quality of such biomarkers is essential, since manipulation of stem cells can unintentionally induce differentiation. Many biomarkers have been identified and validated;however most correspond to protein expression and so require the invasive application of extrinsic labels to cells prior to analysis. We hypothesize and have preliminary data to suggest that intrinsic metabolic signatures (i.e., NADH, FAD) can serve as noninvasive biomarkers of stem cells when detected with multiphoton optical based approaches. Multiphoton imaging analysis of intrinsic biomarkers is advantageous over other fluorescence microscopy methods due to its ability to probe deeply within multicellular aggregates (as is frequently the case with stem cells), compatibility with other advanced optical methods such second harmonic generation imaging (SHG) and fluorescence lifetime imaging (FLIM) and ability to tune over a broad range of excitation wavelengths. We have recently coupled multiphoton optics to a flow cytometry system that can analyze single cell and multicell aggregates in a high throughput manner, with potential to sort cell populations based on optical outputs, including intrinsic fluorescence. Thus we have the potential to rigorously define intrinsic signatures of stem cells and to purify populations based on these signatures, creating an ideal graft for transplantation. We propose to take substantial steps toward this goal via the following aims: Aim 1, to identify robust intrinsic signatures predictive of cardiomyocyte differentiation of human embryonic stem cells (hES) and Aim 2, to purify embryoid bodies based on intrinsic signatures and determine the cardiogenic differentiation potential and functional capacity of such sorted populations. If successful, we will have fulfilled a long unmet need for clinical stem cell transplantation and basic stem cell biology. Perhaps the most immediate benefactors of this project are the over 50,000 bone marrow, cord blood or enriched peripheral blood transplant recipients each year who currently receive a heterogeneous population of mononuclear cells. The composition of these transplants could be optimized if there existed an instrument capable of distinguishing cells without extrinsic markers and capable of sorting cells after identification - both are possibilities with success of this proposal. We will look specifically at cardiomyocyte differentiation here as future cellular therapies under development for the treatment of a broad spectrum of diseases including heart disease will potentially benefit from such detection and cell sorting strategies. PUBLIC HEALTH RELEVANCE: Tens of thousands of bone marrow, cord blood or enriched peripheral blood transplants are performed each year worldwide to treat a myriad of blood cancers. The composition of these transplants could be screened and optimized if there existed non-invasive biomarkers capable of predicting stem cell fate and instrumentation capable of distinguishing and sorting heterogeneous cells based on such biomarkers. If successful, the aims of this project will fulfill these needs.